Randomized clinical trial of astaxanthin supplement on serum inflammatory markers and ER stress‐apoptosis gene expression in PBMCs of women with PCOS

Abstract Polycystic ovarian syndrome (PCOS) is related to pro‐apoptotic and pro‐inflammatory conditions generated by Endoplasmic reticulum (ER) stress. This study aimed to determine the effect of Astaxanthin (ASX), as carotenoid with potent antioxidant and anti‐inflammatory properties, on serum inflammatory markers, apoptotic factors and ER stress‐apoptotic genes in peripheral blood mononuclear cells (PBMCs) of women with PCOS. This randomized, double‐blind clinical trial included 56 PCOS patients aged 18–40. For 8 weeks, subjects were randomly assigned to one of two groups: either 12 mg ASX (n = 28) or placebo (n = 28). Real‐time PCR was used to quantify gene expression associated with ER stress‐apoptosis in PCOS women's PBMCs. The levels of TNF‐α, IL18, IL6 and CRP were determined by obtaining blood samples from all patients before and after the intervention using Enzyme‐linked immunosorbent assay (ELISA). Also, the levels of active caspase‐3 and caspase‐8 were detected in the PBMC by ELISA kit. Furthermore, we evaluated the efficacy of ASX on disease symptoms. Following the 8‐week intervention, ASX supplementation was able to reduce the expression of GRP78 (p = 0.051), CHOP (p = 0.008), XBP1 (p = 0.002), ATF4 (0.038), ATF6 (0.157) and DR5 (0.016) when compared to the placebo. However, this decrease was not statistically significant for ATF6 (p = 0.067) and marginally significant for GRP78 (p = 0.051). The levels of TNF‐α (p = 0.009), IL‐18 (p = 0.003), IL‐6 (p = 0.013) and active caspase‐3 (p = 0.012) were also statistically significant lower in the therapy group. However, there was no significant difference in CRP (p = 0.177) and caspase‐8 (p = 0.491) levels between the treatment and control groups. In our study, ASX had no significant positive effect on BMI, hirsutism, hair loss and regularity of the menstrual cycle. It appears that ASX may benefit PCOS by changing the ER stress‐apoptotic pathway and reducing serum inflammatory markers; however, additional research is required to determine this compound's potential relevance.


| INTRODUC TI ON
Among women of childbearing age, PCOS is the most common endocrine condition and the major cause of infertility. 1 There is a wide variation in the prevalence of this syndrome across countries. 2 may be affected by factors such as racial diversity and physical composition. 3According to the Rotterdam agreement, more than 15% of women have PCOS. 4,5There are numerous characteristics associated with PCOS, such as hyperandrogenism (HA), metabolic disturbances, irregular menstruation, anovulation, hirsutism and infertility. 6There is no clear explanation for the aetiology of PCOS, but genomic and environmental factors are believed to contribute to this condition. 7,8According to recent research, chronic low-grade inflammation has a significant role in the development of PCOS. 9 In fact, as a pro-inflammatory condition, PCOS is associated with cardiovascular disease and type 2 diabetes. 10In numerous investigations comparing women with this syndrome to controls, tumour necrosis factor (TNFα), interleukin-6 (IL-6) and interleukin-18 (IL-18), as well as high-sensitivity C-reactive protein (hs-CRP), were shown to be significantly elevated. 11,12It was observed that there is a positive correlation between TNFα and IR.Additionally, IR may increase the level of IL-6, a known risk factor for cardiovascular disease in females.There is also a correlation between increased levels of IL-1α, IL-1β, IL-2, IL-8, IL-9, IL-15, IL-17, IL-23 and interferonγ (IFNγ) with PCOS.4][15][16] In addition, numerous studies have demonstrated the involvement of apoptotic dysregulation in the development of PCOS.Caspases play a critical role in the process of apoptosis.8][19] Studies have also looked at the connection between PCOS and Endoplasmic reticulum (ER) stress.1][22] ER stress develops as a pathologic condition when unfolded or misfolded proteins accumulate in tissues with excessive protein production.The induction of ER stress can occur under physiological and pathological circumstances such as glucose deprivation, inflammation and oxidative stress (OS), as well as high free fatty acid levels, abnormal Ca 2+ regulation and hypoxia. 23Under ER stress, unfolded protein response (UPR) occurs to protect the cells. 23UPR is regulated by the 78-kDa glucose-regulated protein (GRP78) and the three transmembrane proteins it contains: protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). 24If ER stress cannot be alleviated, the cell undergoes apoptosis. 25It's still not clear how ER stress triggers apoptosis, and the mechanism may vary depending on the stimulus and cell type.Cell apoptosis under ER stress is clearly linked to overexpression of the UPR transcription factor C/EBP homologous protein (CHOP). 257][28] A recent study has demonstrated that human and mouse cumulus cells undergo apoptosis after testosterone-induced ER stress, via induction of DR5. 29 A number of studies have suggested that DRs and caspase-8 may contribute to the promotion of apoptosis subsequent to ER stress. 26,27,30,31It has been shown that, caspase-8 has the ability to initiate the activation of caspase-3 and caspase-7. 32ER stress, on the other hand, exerts a close relationship with inflammation and OS.It has been demonstrated that all three main UPR pathways regulate the transcriptional program that promotes inflammation through transcription factors like NF-ΚB (Nuclear factor-κB) and activator protein-1 (AP-1). 33OS is often associated with inflammation, obesity, insulin resistance (IR) and HA, which are all common symptoms and potential causes.These factors have a strong correlation with OS.In addition, excessive androgen levels can lead to OS, IR and inflammation.The interaction of these factors contributes to the development and worsening of ovulation disorders in PCOS, particularly in individuals who are overweight or obese. 34,35Recently, there has been increasing emphasis on the use of antioxidants as an adjuvant treatment for PCOS.It has been reported that ASX, as a xanthophyll carotenoid found in several microorganisms, has been reported to be a potent antioxidant with no negative effects.It has been found that ASX has anti-inflammatory, antiapoptotic, antioxidative, anticancer, neuroprotective and immunomodulatory effects, among others. 36cording to considerable in vivo and in vitro research, ASX appears to have anti-inflammatory properties in mammals, raising the prospect that it could be a useful treatment for disorders related to inflammation. 379][40] Meanwhile, recent research from our lab suggested that ASX may be able to influence ER stress and OS in the granulosa cells of PCOS patients. 41As OS induces ER stress and plays a pivotal role in the pathogenesis of PCOS, and also considering the interactions between ER stress, inflammation, OS and apoptosis, in this study, we examined the effect of ASX on serum inflammatory markers, active caspase levels and gene expression related to ER stress-apoptosis in PBMC of women with PCOS.Furthermore, we evaluated the efficacy of ASX on disease symptoms.

| Trial design and participants
Between October 2021 and May 2022, 56 PCOS patients aged 18-40, were enrolled in the current randomized, double-blind trial at the Omid Clinic in Tehran, Iran, which is registered at http:// www.irct.ir: IRCT20201029049183N2. Prior to any intervention, all participants signed informed consent forms (ethics committee reference number: IR.TUMS.MEDICINE.REC.1400.1051).Pregnant women and women with metabolic disorders, including thyroid disease, Cushing's syndrome, hyperprolactinemia, diabetes mellitus, congenital adrenal hyperplasia and impaired glucose tolerance, as well as current or particular past diet or physical activity programs (within the last 3 months) were not included in the study.In addition, all of the patients in our research had no medical history of inflammatory or autoimmune diseases.For 8 weeks, participants were randomized into two groups and given either a placebo (n = 28) or 12 mg capsules in terms of colour, shape, size, packaging and other attributes.Astareal Company (Tokyo, Japan) provided ASX capsules and placebos.Checking the capsule containers and sending daily text messages reminding participants to take their supplements and placebos were used to gauge compliance.The compliance was ensured through the use of 24-h food diary records completed during the study.Additionally, a modified version of the Nutritionist IV program, which is based on Iranian foods, was utilized to estimate the dietary intake of patients.In addition, we utilized the Persian version of the 7-item International Physical Activity Questionnaire (IPAQ) to monitor and regulate physical activity levels.The scoring system provided by the IPAQ was used to classify physical activity levels into three categories: inactive, minimally active and healthenhancing physically active.Regarding clinical signs, a 21-35-day menstrual cycle was considered regular.The study also used the Ferriman-Gallwey criteria to determine the hirsutism score, as well as the Ludwig visual score for hair loss.

| Assessment of outcomes
Inflammatory markers were considered as the primary outcomes, and biomarkers of apoptosis, gene expression, clinical features of hyperandrogenism (hirsutism and hair loss), and irregular menstruation cycle were considered as the secondary outcomes.

| Randomization and blinding
The study began with the inclusion of 56 women.We used blocked randomization to assign participants to either a control (placebo) or intervention (ASX) group at random.Participants and researchers were kept in the dark about randomization and allocation to the study.An overview of the study map can be seen in Figure 1.

| Isolation of lymphocyte cells
Density gradient centrifugation with Ficoll-Hypaque (Lymphodex, Inno-Train, Germany) was used to isolate lymphocytes from the blood.It is best to describe the procedure as follows: fresh blood is combined with an equal volume of Phosphate Buffer Saline (PBS), and then carefully layered over Ficoll in a 2:1 ratio.The sample was subjected to 1100 g of centrifugal force for 25 min at 20°C.As a final step, the middle layer was slowly transferred to a fresh falcon and twice washed with PBS at 300 g for 10 min.

| RNA extraction and real-time PCR
The extraction of RNA was done manually.The RNX-plus Solution was utilized in the process of RNA isolation (Cinnacolon, Tehran, Iran).Until cDNA could be made, it was held at −20°C.A spectrophotometer was used to determine the quantity of total RNA extracted from each sample (Biochrom WPA Biowave, Cambridge, UK).Then, we synthesized complementary DNA, using a cDNA synthesis kit (Sinnaclon, Tehran, Iran) according to the manufacturer's instructions.Quantitative RT-PCR was used to evaluate the expression of XBP1, ATF4, ATF6, GRP78, CHOP, DR5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene using Lightcycler 96 System (Roche, Germany) and Real Q Plus 2x Master Mix Green (Amplicon, Denmark).mRNA expression calculations were calculated using the Livak technique (2 −ΔΔCt ) (Livak and Schmittgen, 2001).Table 1 displays the primer sequences.

| Enzyme-linked immunosorbent assay
At the beginning and end of the intervention, 10 mL of venous blood samples were collected to determine serum levels of TNFα, IL-18, IL-6, CRP, caspase-3 and caspase-8.Abcam enzyme-linked immunosorbent assay (ELISA) kit utilized according to the manufacturer's instructions for TNFα, IL-18 and IL-6 (abcam ab181421, ab215539 and ab178013, USA, respectively).In addition, CRP was evaluated using the particle enhanced turbid metric approach in this study (Pishtaz-Teb Diagnostics, Tehran, Iran).Also, we utilized Invitrogen Thermo Fisher Scientific kits (Invitrogen, Camarillo, CA, USA), for detection the levels of caspase-3 (KHO1091) and caspase-8 (BMS2024) in the PBMC, in compliance with the manufacturer's protocols.The results were presented per μg of protein found in PBMC lysates.

| Statistical analysis
The Kolmogorov-Smirnov test was utilized to confirm the parameters' normality, and data were reported as means± SD (standard deviations).In accordance with the proposed formula for parallel clinical trials (Kelsey et al.), and serum CRP levels as a key variable, we calculated a sample size of 25 participants per each group 42 based on type I error of 5% (α = 0.05), type II error of 20% (β = 0.20; power = 80%).Given the anticipated 5% attrition rate, the sample size for each group was determined to be 28 individuals.Statistical analysis was performed using SPSS version 22 with a significance level of p < 0.05.In order to compare the baseline characteristics of the two groups, we utilized a chi-square test for categorical data and an independent sample t-test for continuous data.Also, variables change between the two intervention groups were compared by independent sample t-tests.For intra-group changes, Mcnemar test used for categorical variable.In order to avoid potential bias, analysis of covariance (ANCOVA) was used to adjust for the effects of baseline values (dependent variable) such as BMI and age.The correlations between the serum levels of the cytokines with the signs of diseases and baseline variables were investigated using Pearson's correlation coefficient.

| RE SULTS
In this randomized clinical trial (RCT), 56 participants were first split into two groups of 28.Ultimately, 53 participants completed the trial: 26 in the ASX group and 27 in the placebo group; all dropped out in both groups was for personal reasons (1 and 2 patients in ASX and placebo groups, respectively) (Figure 1).The compliance rate in our study was high, with more than 90% of capsules consumed in both groups during the study.Throughout the trial, no adverse effects were noted in association with the ASX supplement in PCOS women.There were no significant differences between the ASX group and placebo in terms of age, BMI, basal hormones (LH, FSH and Testosterone), disease duration, smoking and alcohol consumption (p > 0.05) (Table 2); this also applies to intake of nutrients (Table 3).
About physical activity, all of the patients in our study engaged in minimally active category.In terms of inflammatory cytokines, IL-18, IL-6, TNFα and CRP, there were no statistically significant differences between the two groups at the baseline (p > 0.05) (Table 4).Table 4 indicated that, ASX treatment was related to a significant drop in IL-18 (p = 0.003), IL-6 (p = 0.013) and TNFα (p = 0.009) versus to placebo.
Additionally, there was a reduction in the levels of active caspase-3 (p = 0.012) and caspase-8 (p = 0.491) in the treatment group; however, this reduction was statistically significant only in caspase-3 (Figure 3).
In Table 5, it is evident that there were no significant differences between the two groups at the baseline in terms of normal menstrual cycle, hirsutism and hair loss.The study found that there was a slight improvement in menstrual cycle irregularity in both the ASX and placebo groups after intervention.However, this improvement was not statistically significant (p = 0.386).The results indicated that, normal menstrual cycle increased after ASX therapy in the treatment group compared to the baseline values (p = 0.031).In addition, no significant changes were observed in hirsutism (p = 0.398) and hair loss (p = 0.784) in both groups.that serum CRP correlated positively with hirsutism (r s = 0.343; p = 0.012) and hair loss (r s = 0.245; p= 0.047).However, no significant correlation was shown between other variables.

| DISCUSS ION
This RCT examines the effects of ASX consumption on the ER stress-apoptosis pathway and serum inflammatory markers in PCOS women.The result indicated in PCOS women, 8 weeks of oral treatment of 12 mg ASX decreased serum levels of TNFα, IL-18, IL-6 and CRP.In the treatment group mRNA expression levels of CHOP, ATF4, XBP1 and DR5 decreased compared to the placebo group; however, this reduction was not statistically significant in ATF6, and it also had a borderline effect on GRP78.Also, ASX had no significant positive effect on BMI, hirsutism, hair loss and regularity of the menstrual cycle in women with PCOS.A persistent low-grade inflammation condition has been reported in patients with PCOS, including elevated leukocyte numbers and dysfunction of pro-inflammatory cytokines. 43Cardiovascular disease and Type 2 diabetes mellitus (T2DM) have been associated with this inflammatory condition. 44ere is a correlation between chronic inflammatory processes and elevated levels of inflammatory cytokines, such as IL-1β, TNFα and IL-6. 45,46In addition, it has been reported that, PCOS has been linked to greater levels of hsCRP, TNFα, IL-6 and IL-18 compared in healthy controls. 11,12,47High levels of IL-18 in PCOS patients were found to be correlated with insulin sensitivity and obesity. 45It has been observed that IL-18 induces TNFα, resulting in the production of IL-6. 480][51] IL-6 and TNFα stimulate the liver to produce CRP.CRP is commonly used to measure chronic low-grade inflammation in PCOS studies.A growing body of research suggests that CRP is a biomarker of intravascular inflammation and a key indicator of cardiovascular disease. 52Elevated levels of CRP may potentially elucidate the heightened susceptibility of some patients with PCOS to the beginning of cardiovascular disease (CVD) at an earlier age. 53ASX is a potent antioxidant that effectively inhibits the initiation of inflammation in various biological systems. 54According to previous research, ASX has been shown to reduce inflammatory cytokine expression and modify inflammatory signalling pathways. 557][58][59] Also, according to a recent review, ASX may be a promising treatment option for chronic inflammatory illnesses and may also protect against skin and gastrointestinal ailments and vascular stiffness by controlling inflammation. 55Similar to findings from prior studies, our investigation found that the levels of TNFα, IL-18 and IL-6 reduced dramatically in the therapy group, it was while the reduction in CRP levels was comparable.Wei Xia et al. discovered in a systematic review and meta-analysis that taking ASX was associated with a decrease in CRP levels.This association was noticed when ASX was administered for at least 12 weeks or at high doses above 12 mg/day. 60It appears that if ASX had been supplied at a higher dose and for a longer duration in our trial, the CRP reduction would have been significant.
Excessive ER stress has been implicated in the aetiology of PCOS and is thought to contribute to cell death.Studies have b Based on linear mixed effects model (the included variables were: basic value of dependent variable, treatment type, Age and BMI).
c Statistically significant.
shown that ER stress inhibitors can ameliorate many PCOS symptoms, which may be a novel entry point for treating PCOS. 61The ER protein homeostasis is controlled by the UPR mechanism.
While in normal settings, the UPR is not engaged, it can be activated in the presence of certain stressors. 62GRP78 is an essential in maintaining the homeostasis of the ER. 39During pathological conditions, GRP78 activates the UPR pathway by releasing three ER stress sensors.Due to this, the degree of ER stress is closely correlated with the expression of GRP78.ATF6, ATF4, GRP78 and CHOP. 39Similarly, in another study, Selim Demir et al. found that in the testicular TID model, high levels of ER stress cause testicular tissue damage and that ASX prevents this damage by decreasing the level of ER stress. 65A number of studies have suggested that DRs and caspase-8 may contribute to the promotion of apoptosis subsequent to ER stress. 26,27,30,31 has been shown that, caspase-8 has the ability to initiate the activation of caspase-3 and caspase-7. 32A growing body of evidence supports a strong correlation between the therapeutic effects of ASX and its anti-apoptotic properties. 66In our prior in- From our perspective, the small sample size is a significant limitation of the present study.Perhaps using a larger sample size in the study would have resulted in statistically significant clinical changes.
The results of this investigation suggest that a positive correlation exists between CRP levels and clinical manifestations of HA (hirsutism and hair loss).However, no significant correlation was shown between other variables.A research study by Nehir et al.
demonstrated a positive correlation between FAI and CRP, TNFα and α-1 glycoprotein. 71Additionally, prior research has shown a link between inflammatory indicators and HA in PCOS. 72,73At present, there is no clarity regarding whether inflammation induces HA by stimulating theca cell androgen synthesis, or whether HA itself initiates the inflammatory response.Some research on PCOS patients suggests that androgens can stimulate inflammatory cells and initiate the inflammatory process.On the other hand, some experts believe that chronic inflammation is the primary factor behind androgen production and ovarian dysfunction. 74e remarkable component of our study is the inclusion of all PCOS phenotypes, which considerably increases the possibility of generating data that can be generalized to a larger population.

R E FE R E N C E S
24 ATF4 serves as another indicator of ER stress; it has been shown that ATF4 induces CHOP expression under ER stress.CHOP increases levels of pro-apoptotic molecules such as DR5 in response to extensive or persisted ER stress. 62ATF6, as another branch of UPR, stimulates protein folding and degradation pathways associated with the ER in a manner analogous to IRE1-XBP1. 63ER stress, on the other hand, exerts a close relationship with inflammation and OS.It has been demonstrated that all three main UPR pathways regulate the transcriptional program that promotes inflammation F I G U R E 2 The fold changes levels of CHOP (A), ATF4 (B), XBP1 (C), DR5 (D), ATF6 (E) and GRP78 (F) in pbmcs of placebo and treatment groups.Statistical significance (p < 0.05) was assessed by t-test.Differences between groups; *p < 0.05, **p < 0.01 and ***p < 0.001.F I G U R E 3 Protein concentrations of active caspase-3 and caspase-8 measured using ELISA in PBMCs of placebo and treatment groups.Statistical significance (p < 0.05) was assessed by t-test.Differences between groups; *p < 0.05.through transcription factors like NF-ΚB and AP-1. 33In the present study, we determined the effect of ASX on ER stress-apoptosis in the PBMCs of PCOS patients; the results demonstrated the expression levels of CHOP, GRP78, XBP1, ATF4, ATF6 and DR5 mRNA decreased compared to the placebo group.Similar to our findings, Bhuvaneswari et al. found that ASX decreases the levels of ATF6 and PERK, which preserve liver tissue from the harmful effects of a high fat and fructose diet 64 ; on the other hand, it has been shown that ASX attenuated brain injury by reducing levels of GRP78 and CHOP. 40Recent research by Wang et al. demonstrated that administration of ASX prevents mice from developing ethanol-induced cardiomyopathy by lowering their levels of PERK,

However, it is
important to acknowledge that the present study does possess several limitations.Initially, there was a lack of objective measures, such as the quantification of ASX levels in serum or plasma, which made it difficult to evaluate patient compliance The present analysis yielded findings that indicate the potential influence of ASX on the modulation of gene expression related to the ER stress pathway.Nevertheless, it is imperative to assess posttranslation alterations and various other aspects.Ultimately, it is conceivable that the comparatively brief duration of the intervention could have played a role in the development of insignificant results.Hence, it is imperative to undertake further study over an extended duration and with larger sample sizes in order to validate our findings.In conclusion, our findings show that 8 weeks ASX administration in PCOS patients can modulate ER stress-apoptosis in PBMCs by changing the expression of genes implicated in the UPR process and apoptosis.ASX, on the other hand, reduced serum levels of proinflammatory markers, such as IL-18, TNFα, IL-6 and CRP in PCOS patients.More extensive research is required to determine the potential role of ASX.

Table 6 ,
serum levels of IL-18, IL-6, TNFα and CRP were analysed for correlations.Correlation analysis showed TA B L E 1 Forward and reverse primers used for real-time quantitative PCR.Baseline characteristics of study participants.Dietary intake of study participants throughout the study.
Comparison of serum inflammatory markers between the two groups.
67stigation, we demonstrated that the intake of ASX by infertile women with PCOS resulted in improved levels of apoptotic factors in both serum and follicular fluid, as well as modulation in gene and protein expression related to the apoptosis pathway in granulosa Based on Chi-square.Correlations between pro-inflammatory cytokines and Baseline parameters. .6n the present study, in line with recent studies, we found that, the levels of caspase-8 and caspase-3 reduced in the therapy group, however this reduction was comparable in caspase-8.
a Based on Mcnemar test.b cellsgroups.There may be several factors that contribute to the variations between our study and other studies, including the type and amount of supplement, participant characteristics (such as age, genetics and body measurements), pharmacokinetic factors, the duration of the participants' follow-up, and the number of participants.